Defensins: Transcriptional regulation and function beyond antimicrobial activity

Journal Pre-proof Defensins: Transcriptional regulation and function beyond antimicrobial activity Gabriela Contreras, Iman Shirdel, Markus Santhosh Braun, Michael Wink PII:

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https://doi.org/10.1016/j.dci.2019.103556

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DCI 103556

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Developmental and Comparative Immunology

Received Date: 26 August 2019 Revised Date:

13 November 2019

Accepted Date: 15 November 2019

Please cite this article as: Contreras, G., Shirdel, I., Braun, M.S., Wink, M., Defensins: Transcriptional regulation and function beyond antimicrobial activity, Developmental and Comparative Immunology (2019), doi: https://doi.org/10.1016/j.dci.2019.103556. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Defensins: Transcriptional regulation and function beyond antimicrobial activity

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Gabriela Contreras1, Iman Shirdel2, Markus Santhosh Braun1, Michael Wink1

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1

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Heidelberg, Germany

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2

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Corresponding author:

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Michael Wink, e-mail address: [email protected]

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Postal address: Institute of Pharmacy and Molecular Biotechnology, Heidelberg

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University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany

Institute of Pharmacy and Molecular Biotechnology, Heidelberg University,

Marine Sciences Faculty, Tarbiat Modares University, Noor, Iran.

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Gabriela Contreras, e-mail address: [email protected]

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Postal address: Institute of Pharmacy and Molecular Biotechnology, Heidelberg

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University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany

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Abstract

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Defensins are one the largest group of antimicrobial peptides and are part of the innate

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defence. Defensins are produced by animals, plants and fungi. In animals and plants,

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defensins can be constitutively or differentially expressed both locally or systemically

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which confer defence before and a stronger response after infection.

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Immune signalling pathways regulate the gene expression of defensins. These pathways

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include cellular receptors, which recognise pathogen-associated molecular patterns and

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are found both in plants and animals. After recognition, signalling pathways and,

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subsequently, transcriptional factors are activated.

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There is an increasing number of novel functions in defensins, such as

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immunomodulators and immune cell attractors. Identification of defensin triggers could

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help us to elucidate other new functions. The present article reviews the different

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elicitors of defensins with a main focus on human, fish and marine invertebrate

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defensins.

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Keywords: Host defence peptides, defence response, immunity, defensins, innate

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immune response, gene expression.

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Abbreviations:

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AMPs: antimicrobial peptides, CSαβ: cysteine-stabilised αβ, HBD: human β-defensins,

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HD: human defensin, HNPs: human neutrophil peptides, NF-κB: nuclear factor-κB,

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PAMPs: Pathogen-associated molecular pattern, ROS: reactive oxygen species, PRR:

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pattern recognition receptors, TLRs: Toll-like receptors. 1

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1. Introduction

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Organisms have developed different defence strategies to eliminate pathogens and for

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protection of damaged tissues. The first line of defence, so-called innate immunity,

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include mechanical barriers, such as the presence of an epithelium, mucus secretion in

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animals, and bark, waxy cuticular layers and trichomes in plants. Additionally, a

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chemical barrier composed of secondary metabolites, reactive oxygen species (ROS),

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nitric oxide, large antimicrobial proteins (e.g., lysozyme and lactoferrin) and

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antimicrobial peptides (AMPs) has evolved.

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AMPs are antimicrobial compounds, naturally synthesised by both eukaryotes and

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prokaryotes. In animals, they are mostly produced by epithelial cells of skin, airways

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and gastrointestinal tract (Zasloff, 2002). In prokaryotes and lower eukaryotes, the role

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of AMPs is less clear. AMPs might help these organisms to compete for nutrients with

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other microorganisms (Ageitos et al., 2017). AMPs are composed of 12 to 50 amino

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acids, and they are commonly cationic and amphipathic. Cationic AMPs interact with

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the negatively-charged bacterial membrane, promoting leakage or its disruption, and

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consequently, bacterial death (Reddy et al., 2004).

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Defensins are one of the best-described groups of AMPs which are found in animals,

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plants and fungi (Figure 1). They are short peptides (18-45 amino acids) and mainly

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positively charged. Defensins form three to six disulphide bridges and have an

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amphipathic structure. The main secondary structure of defensins is a β-hairpin motif;

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however, also they can contain an α-helix.. Defensins have a broad spectrum of

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antimicrobial activity against various pathogens, including both gram-negative and

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gram-positive bacteria, fungi, protozoa and enveloped viruses (Aley et al., 1994; Daher

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et al., 1986; Selsted et al., 1985a). So far, 335 defensins have been described (Wang et

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al., 2015a).

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In vertebrates, defensins are divided into three classes: α-, β- and θ-defensins (Figure

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2A, B and C). These defensins differ in the pairing of disulphide bridges (Ganz, 2003).

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α- and β-defensins are composed of at least a triple-stranded β-sheet. α- Defensins are

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produced by mammals and form three disulphide bridges linked between Cys1-Cys6,

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Cys2-Cys4 and Cys3-Cys5 (Zhao et al., 2016). In humans, they are produced by

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neutrophils (human neutrophil peptides, HNPs) and Paneth cells which are located at

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the base of small intestinal crypts of Lieberkuhn (human defensin [HD] -5 and -6)

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(Ganz et al., 1985; Jones and Bevins, 1992, 1993). 2

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β-Defensins are found in a wide range of vertebrates (Harwig et al., 1994; Meng et al.,

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2013a; Zou et al., 2007). Human β-defensins (HBD) are produced in the airway

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epithelial tract, keratinocytes and monocytes (Bensch et al., 1995; Duits et al., 2002). β-

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Defensins form disulphide bridges between Cys1-Cys5, Cys2-Cys4 and Cys3-Cys6, and

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several of them form an α-helix at the N-terminus (Sawai et al., 2001; Tu et al., 2015).

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Regarding θ-defensins, they are cyclic and derived from two truncated α-defensins.

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They have been identified in some non-human primates, and the disulphide bridges are

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formed between Cys1-Cys6, Cys2-Cys5 and Cys3-Cys4 (Figure 2C) (Tran et al., 2002).

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Defensins, produced by higher plants, fungi and invertebrates, are so-called defensin-

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like peptides (Figure 2D). Many defensin-like peptides are usually composed of an α-

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helix linked to two antiparallel β-sheets by disulphide bridges. This motif is called

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cysteine-stabilised αβ (CSαβ) (Zhao et al., 2016). CSαβ defensins have been classified

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into three groups based on their sequence, structure and functional similarity. These

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groups are antibacterial ancient invertebrate-type defensins (AITDs) which include

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defensins from chelicerates and fungi, antibacterial classical insect type defensin

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(CIRSs) and antifungal plant/insect type defensin (PITDs) (Zhu, 2008).

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In animals and plants, defensins are constitutively or differentially expressed both

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locally and systemically (Harder et al., 1997; Zhao et al., 1996). Various stress factors

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activate defence response pathways which induce the gene expression of defensins and

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other AMPs. Stress factors can be both biotic (e.g., microbial infection) and, in plants,

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abiotic (e.g., dryness, high salinity).

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In addition, there is an increasing number of novel functions in defensins beyond the

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antimicrobial activity. Identification of defensin regulators could help us to elucidate

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other alternative functions. The present article reviews the different elicitors of

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defensins with a main focus on human, fish and marine invertebrate defensins.

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Furthermore, this review presents a general overview of signalling pathways that trigger

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the expression of defensins in vertebrates, invertebrates, fungi and plants.

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2. Transcriptional regulation of defensins

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2.1. Animals

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In animals, microbial infection triggers a defence response which involves the

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production of defensins and other AMPs, conferring protection against infection. This

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response is part of the innate immunity. When pathogens contact epithelia or enter the

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circulatory system, they are recognised by receptors located in epithelial barrier cells, 3

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macrophages, dendritic cells and mast cells. Recognition of microbial products can

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regulate the nuclear factor-κB (NF-κB) signalling pathway. This pathway is conserved

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in animals and has been found in simple organisms such as cnidarians, sea anemones

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and sponges (Gilmore and Wolenski, 2012). Signalling pathways of vertebrate and

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invertebrate defensins share some similarities, that we will discuss later.

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2.1.1. Vertebrates

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In vertebrates, the expression of defensins has mostly been studied in mammals (Figure

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3A). First, pathogen-associated molecular patterns (PAMPs) are recognised via pattern

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recognition receptors (PRR), such as Toll-like receptors (TLRs), Nucleotide

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oligomerisation domain (NOD)-like receptors (NLR), C-type lectin receptor (CLR) and

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Cytosolic DNA sensor (CDS) (Akira and Takeda, 2004; Fritz et al., 2006; Takaoka et

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al., 2007; Wintergerst et al., 1989). PAMPs are characteristic conserved molecules

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within a class of microorganisms, such as lipopolysaccharide (LPS), bacterial DNA

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(unmethylated CpG DNA), β-glucans, flagellin and peptidoglycans. TLRs are

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homologous to Toll receptor from Drosophila (Medzhitov et al., 1997). Ten members

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of TLRs have been described in mammals (reviewed in Medzhitov, 2001). NLRs, such

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as NOD1 and NOD2, are intracellular receptors that recognise bacterial peptidoglycan.

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TLR and NLR recognise PAMPs through an extracellular leucine-rich repeat (LRR)

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domain located at the carboxyl-terminus (reviewed in Becker and O’Neill, 2007). TLRs,

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additionally, possess an intracellular protein-protein interaction domain, TIR (Toll-

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interleukin 1 receptor).

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TLRs transduce the signal through adaptors, such as myeloid differentiation factor 88

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(MyD88), TIR domain-containing adaptor protein (TIRAP), Toll-receptor associated

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molecule (TRAM) and Toll-receptor-associated activator of interferon (TRIF)

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(reviewed in Akira and Takeda, 2004). These adaptors propagate the signal which leads

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to the activation of NF-κB proteins and involves the participation of IRAK, TRAF,

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NEMO and IKK. NF-κB is a family of dimeric transcriptional factors which regulates

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the expression of genes related to the immune response, inflammation process,

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development and control of apoptosis (Gilmore and Wolenski, 2012). In unstimulated

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cells, NF-κB dimer is sequestered in the cytoplasm by the inhibitor of NF-κB (IκB).

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When NF-κB is activated, IκB is ubiquitinated and subsequently degraded, and NF-κB

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is nuclear translocated. IκB contain 5-7 ankyrin repeat domains that mask the nuclear

4

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localisation signal of NF-κB proteins (Li et al., 2006). NF-κB signalling pathway has

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been revised in Gilmore and Wolenski (2012), Lawrence (2009) and Moynagh (2005).

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In addition to PAMPs, also cytokines induce the expression of defensins (Pioli et al.,

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2006). In mammals, cytokines, such as interleukins (e.g., IL-1β, IL-1α,) and tumour

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necrosis factors (e.g., TNFα) are produced by macrophages, dendritic cells and mast

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cells (Turner et al., 2014). In adaptive immunity, helper lymphocytes are the primary

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source of cytokines. Cytokines bind to receptors, such as IL-1R and IL-17R. This

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interaction triggers a response that includes the activation of the NF-κB pathway. An

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overview of factors that upregulate human defensin gene expression are listed in Table

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1.

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Like human and mammalian counterparts, fish β-defensins can be produced in response

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to various stimuli including bacteria, viruses, bacterial components, viral mimics and

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even algae. A complete list of tissue distribution and the response to various inducers in

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the gene expression of fish β-defensins is presented in Table 2. The presence of multiple

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potential transcription factor binding sites in the upstream promoter region of β-defensin

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genes in fish might confirm that defensin genes are induced by a variety of stimuli

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(Katzenback, 2015). For example, pituitary-specific POU domain transcription factor 1a

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(POU1F1a) might regulate the expression of a β-defensin gene from orange-spotted

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grouper (Epinephelus coioides) (Jin et al., 2010b); and the promoter region of medaka

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β-defensin (Oryzias latipes) contains binding sites of Sp-1 and NF-κB transcription

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factors (Zhao et al., 2009). Overall, limited studies on transcription factors related to

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fish β-defensins have been carried out. However, these studies reveal the similarity and

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conservation of transcription factors in fish and mammals, since the above-mentioned

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transcription factors are also present in mammals.

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2.1.1.1. Other elicitors of mammal defensin gene expression

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Other molecules, distinct to PAMPs and cytokines, have been shown to enhance the

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gene expression of human defensins. These include plant secondary metabolites.

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Theaflavin derivatives induce the secretion of HBD-1, 2 and 4 (Bedran et al., 2015).

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Andrographolide, oridonin and isoliquiritigenin (secondary metabolites of plants used in

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Chinese traditional medicine) induce the HBD-3 gene expression in human colonic

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epithelial cells without activation of the NF-κB pathway and through the activation of

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the EGF receptor and MAPK signalling. This finding suggests that small molecules can

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induce defensins expression avoiding inflammatory response (Sechet et al., 2018).

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Moreover, vitamin D (1,25-dihydroxyvitamin D3) upregulates HNP1-3 and HBD-2 5

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(Subramanian et al., 2017; Wang et al., 2004). On the other hand, ultraviolet A, B and C

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rays upregulate HBDs gene expression (Cruz Díaz et al., 2015; Seo et al., 2001).

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Furthermore, ascorbic acid (vitamin C) up-regulates HBD-1 (Cruz Díaz et al., 2015).

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Other elicitors of HBDs are reviewed in de Prado Montes de Oca (2013).

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2.1.2. Invertebrates

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To respond to pathogen infection and modulate innate immunity response, invertebrates

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possess two independent signal cascades that activate NF-κB transcription factors. For

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invertebrates, the most studied model system of the immune response is Drosophila

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melanogaster. Two innate signalling pathways, against bacteria and fungi, act largely in

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D. melanogaster, Toll signalling and Immune deficiency (IMD) pathway (Hedengren-

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Olcott et al., 2004) (Figure 3B). Toll signalling mainly responds to Gram-positive

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bacteria and fungi. First, Toll receptor is activated by Spaetzle, and his interaction

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triggers a cascade that activates Dorsal and Dorsal-like immune factor (DIF) (Ip et al.,

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1993; Lemaitre et al., 1996). The inactivated form of Dorsal/DIF is retained in the

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cytoplasm by Cactus (an IkB homolog). Cactus phosphorylation triggers the

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degradation and nuclear translocation of DIF (Geisler et al., 1992).

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IMD pathway is mainly directed against Gram-negative bacteria. Peptidoglycan

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activates the membrane receptor Peptidoglycan Recognition Protein-LC (PGRP-LC).

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This interaction enables IMD, which interacts to TAK and this one to IKK, stimulating

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Relish (Lemaitre et al., 1995). Relish, DIF and Dorsal are transcriptional factors,

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members of the NF-κB protein family (Dushay et al., 1996; Han and Ip, 1999;

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Rutschmann et al., 2000). Immune response pathways in Drosophila has been revised in

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Engström (1999) and Khush et al. (2001).

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In addition to Toll signalling and IMD pathway, other signalling pathways regulate the

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innate immunity in Drosophila, such as the JAK-STAT pathway. This signalling

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pathway involves transmembrane receptors, Janus kinases (JAKs), and signal

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transducers and activators of transcription (STATs) (reviewed in Arbouzova and

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Zeidler, 2006). JAK-STAT and Toll pathway also regulate developmental processes in

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Drosophila (Arbouzova and Zeidler, 2006; Govind, 2008).

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2.1.2.1. Other elicitors of invertebrate defensin gene expression

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In addition to PAMPs, other factors regulate defensin production, such as 20-

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hydroxyecdysone, a steroid hormone-related to development, ecdysis, reproduction, 6

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apoptosis and immune response (Beckstead et al., 2005; Han et al., 2017). In

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Drosophila, starvation and ionising radiation up-regulate the gene expression of

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drosomycin, a defensin with antifungal activity (Moskalev et al., 2015). An overview of

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factors that regulates invertebrate defensin gene expression are listed in Table 3.

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2.2. Plants

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To detect the presence of pathogens and trigger a defence response, plants possess PRR.

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Some of these receptors are nucleotide-binding site plus leucine-rich repeats (NBS-

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LRR), cell surface receptor-like transmembrane proteins (RLP) and receptor-like

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kinases (RLK) (Zipfel, 2008). Many of these receptors contain LRR, as mammalian

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TLRs. Moreover, the domain at the N-terminus of some NBS-LRR possesses homology

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to TIR, the cytoplasmic signalling domain of Toll receptor of Drosophila and IL-1R of

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mammals (Deslandes et al., 2002; Whitham et al., 1994). Nonetheless, no adaptors

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analogous to MyD88 have been identified in plants.

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Recognition of MAMPs (microbe-associated molecular pattern) activates the mitogen-

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activated protein kinase (MAPK) cascade, and this, in turn, induces the expression of

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defensins and other AMPs (Meng et al., 2013b) (Figure 3C). This response is named

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MAMP-triggered immunity (MTI).

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Microorganisms can suppress MTI by sending effector proteins (i.e., avirulence factors,

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Avr) to interfere with the defence response. In these circumstances, plants develop

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resistance proteins (R proteins) to detect the presence of Avr and trigger a stronger

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response. Defence response mediated by R proteins is called effector-triggered

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immunity (ETI). ETI involves the activation of MAPK signalling pathway.

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MAPK signallings are conserved pathways found in eukaryotes. MAPK signalling

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pathways comprise MEKK, MKK4/MKK5 and MPK3/MPK6. MAPK signalling

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activates WRKY transcriptional factors which are related to the innate immune response

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(Asai et al., 2002). These transcriptional factors have not been found in animals

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(Ausubel, 2005). Involving of MAPK signalling pathway in the plant defence response

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is revised in Taj et al. (2010).

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Similarities in immune response in animals and plants, such as the presence of receptors

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containing TIR and LRR domain, suggest a convergent evolution and not a common

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origin (Ausubel, 2005).

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In the defence response of plants, other cellular messengers participate, such as nitric

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oxide, ROS and plant hormones. Hormones play a crucial role in biotic stress. Under

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MTI and ETI, plants produce salicylic acid, ethylene and jasmonic acid. These 7

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hormones activate signalling cascades whose response includes the expression of

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defensins as well as other AMPs and plant secondary metabolites. For example, in A.

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thaliana, the defence response against fungal infection include the jasmonic acid and

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ethylene signalling pathways. These pathways activate the transcriptional factors,

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ORA59 and ERF1, which regulate the expression of PDF1.2 (Pré et al., 2008). Further

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information regarding the participation of plant hormones in defence response is revised

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in Shigenaga and Argueso (2016).

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For defence, plants produce a mixture of secondary metabolites, such as alkaloids,

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phenolics, terpenes, that attack multiple molecular targets. They are mostly

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constitutively expressed but can be further enhanced by stress. Further information on

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plant secondary metabolites is reviewed in Wink (2008, 2015).

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Apart from biotic stresses (wounding, infection and herbivore attack), abiotic stresses

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(cold, high salinity and dehydration) also regulate expression of plant defensins,

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(Penninckx et al., 1996; Weerawanich et al., 2018). Some defensins and their inducers

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are listed in Table 4.

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2.3. Fungi

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It has been proposed that in fungi, AMPs confer an ecological advantage over nutrient

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competitors (Meyer et al., 2005). In agreement with that, gene expression of some

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fungal defensins is altered by environmental stress conditions (Garrigues et al., 2016;

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Meyer and Stahl, 2002; Paege et al., 2016).

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For CSαβ defensins, regulation of their gene expression has been partly studied in few

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members, anisin1 and aclasin, which are produced by Aspergillus nidulans and

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Aspergillus clavatus, respectively (Contreras et al., 2019; Eigentler et al., 2012). Anisin

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has been related to conidiation (asexual development), and it is involved in oxidative

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stress signalling (Eigentler et al., 2012). Aclasin showed increased expression under

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oxidative conditions and the presence of Bacillus megaterium (Contreras et al., 2019).

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Yeast also possess MAPK cascades, which are involved in the mating pheromone

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response, cell integrity, high osmolarity growth and filamentation (revised in Gustin et

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al. 1998). However, it has not been involved in the immune response. Also, it is not

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known whether any PRR receptor recognises bacteria and fungi through PAMPs as in

265

plant and animals.

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3. Tissue localisation, characterization and functions

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3.1. Animals 8

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3.1.1. Vertebrates

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Vertebrate defensins have been found in mammals, birds, reptiles, fishes and

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amphibians. They are predominately found in epithelial cells located in skin

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(keratinocytes), respiratory, reproductive and gastrointestinal tract (Paneth cells)

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(Diamond et al., 1991; Jones and Bevins, 1992). Moreover, defensins are produced by

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phagocytic cells, such as neutrophils, macrophages and monocytes (Figure 4A) (Ganz et

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al., 1985; Selsted et al., 1985b). The tissue localisation of defensins varies among

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species. β-defensins are the primary group among vertebrate defensins, and they form

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the largest group of defensin families (Qi et al., 2016). α- and θ-defensin families have

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been found only in mammals. These groups of defensins evolved from an ancestral β-

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defensin gene, as results of duplication (Semple et al., 2003).

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Mammal defensins have been described from primates, mice, rabbits, horses, dogs and

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pigs (Aono et al., 2006; Couto et al., 1992; Nagaoka et al., 1991; Sang et al., 2005).

282

They

283

(Krisanaprakornkit et al., 1998; O’Neil et al., 1999). Apart from epithelial cells,

284

mammals defensins are also found in cells related to defence against microbial

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infection, such as macrophages, neutrophils and Paneth cells (Ganz, 2003).

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Defensins have other functions alternative to antimicrobial activity. In vertebrates, some

287

defensins induce the degranulation of mast cells (Befus et al., 1999; Niyonsaba et al.,

288

2001). Moreover, it has been reported that HBD-2, HBD-3 and HBD-4 induce the

289

expression of cytokines in human keratinocytes, macrophages and monocytes

290

(Funderburg et al., 2011; Jin et al., 2010a; Niyonsaba et al., 2007). HBD-3 suppresses

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induction of TNF-α and IL-6 in the presence of lipopolysaccharide when it is at a basal

292

level (Semple et al., 2010). Defensins also participate in the adaptive immune response.

293

Some defensins induce chemoattraction in macrophages, immature dendritic cells and

294

memory T cells (Chertov et al., 1996; Yang et al., 1999). Other functions of vertebrate

295

defensins are revised in Yang et al. (2002).

296

Just like in mammals, a large number of defensins have been identified in birds (e.g. in

297

chicken, zebra finches, turkeys, ostriches, penguins, and ducks) (reviewed in van Dijk et

298

al., 2008). Avian defensins are β-defensins (AvBD) containing triple-stranded β-sheets

299

and thereby resemble their mammalian counterparts. They are of myeloid or epithelial

300

origin and are expressed in various tissues, such as reproductive tract, respiratory tract,

301

digestive tract, bone marrow, heterophils (equivalents to mammalian neutrophils) and

302

skin (reviewed in van Dijk et al., 2008; Zhang and Sunkara, 2014). 9

are

found

in

keratinocytes,

gingival

and

intestinal

epithelial

cells

303

Also, some β-defensin-related peptides have been found exclusively in birds and

304

reptiles (Chattopadhyay et al., 2006; Zhang et al., 2019). Due to their enhanced

305

expression in the oviduct and abundance in egg white, these peptides are referred to as

306

ovodefensins (Gong et al., 2010). Ovodefensins could function as β-defensins and

307

probably play a role in immune response, as they have been found to inhibit the

308

bacterial growth of Escherichia coli (Yu et al., 2018). Phylogenetic studies of

309

ovodefensins of different species suggest that they share a common ancestor and that

310

numerous independent gene duplications occurred after species divergence (Zhang et

311

al., 2019). Ovodefensins contain a distinct spacing pattern in their six-cysteine motif

312

allowing their classification into different subfamilies (Gong et al., 2010; Whenham et

313

al., 2015).

314

Among defensin families, so far only β-defensin has been identified in fish species.

315

Unlike mammals, fish do not have highly developed adaptive immune system and are

316

more dependent on innate immunity. As components of the innate immune system,

317

AMPs play important roles in fish (Chen et al., 2013). In mammals, β-defensin is often

318

expressed in tissues contacting with surrounding environments such as the respiratory

319

tract and gastrointestinal tract which are the first defence lines against pathogens.

320

However, in fish, β-defensin is produced in various tissues, such as in skin, gill,

321

intestine, liver and head kidney (analogous to the mammalian adrenal gland) (Zhu et al.,

322

2017). The presence of a negatively-charged glutamic acid residue, usually at the

323

beginning of β-strand 2, is a specific characteristic of fish β-defensins (Zou et al., 2007).

324

β-defensin peptides in different fish species have typical properties of vertebrates β-

325

defensin. These characteristics include net positive charge, small size, and three

326

disulfide bonds in the mature peptide (Zou et al., 2007). In some fishes, such as olive

327

flounder, mature β-defensin peptide has anionic nature (Nam et al., 2010). Fish β-

328

defensins are structurally and functionally similar to mammalian β-defensins (Masso-

329

Silva and Diamond, 2014), specifically, they are most closely related to HBD-4. Fish

330

defensins exhibit antibacterial activity, antiviral activity, stimulation of chemotaxis and

331

phagocytic activity in leucocytes, as well as immune modulation (Cuesta et al., 2011;

332

Falco et al., 2008; Ruangsri et al., 2013).

333

3.1.2. Invertebrates

334

Invertebrates are exclusively dependent on innate immunity and they do not have

335

acquired immunity. Antimicrobial peptides, particularly defensins, are one of the

336

essential elements in the innate immune system of invertebrates (Yang et al., 2016). 10

337

Invertebrate defensins have been isolated from insects, molluscs, crustaceans and

338

arachnids (Pisuttharachai et al., 2009; Yao et al., 2019; Zhang et al., 2015). They are

339

characterised by the presence of a CSαβ motif, and they contain three or four disulfide

340

bridges.

341

Many insect defensins are produced in the fat body (equivalent to the mammalian liver)

342

after bacterial infection and secreted into the haemolymph. Some defensins of

343

hematophagous insects are produced in the midgut epithelium and are secreted into the

344

gut lumen (Hamilton et al., 2002; Nakajima et al., 2001). Also, insect defensins are

345

expressed in epithelial tissues exposed to the external environment, such as respiratory

346

and reproductive tracts to fight against infections at local level (Ferrandon et al., 1998).

347

Most of the invertebrate defensins are more active against gram-positive than gram-

348

negative bacteria. However, there are few cases, such as drosomycin from Drosophila

349

melanogaster,

350

Pseudacanthothermes spiniger and gallerimycin from Galleria mellonella which exhibit

351

antifungal activity (Fehlbaum et al., 1994; Lamberty et al., 1999; Lamberty et al., 2001;

352

Schuhmann et al., 2003). Besides, drosomycin exhibit antiparasitic activity (Tian et al.,

353

2008).

354

Big defensins are a distinct group from defensins and found only in marine invertebrates

355

(Wang et al., 2014; Wang et al., 2018). A big defensin was identified initially in

356

horseshoe crab (Tachypleus tridentatus), and later in amphioxus, mussel, oyster and

357

scallop (Gerdol et al., 2012; González et al., 2017; Rosa et al., 2011; Saito et al., 1995;

358

Teng et al., 2012; Zhao et al., 2007). Big defensins have limited distribution and have

359

not been found in crustaceans and insects (Gerdol et al., 2012). The size of big

360

defensins is approximate twice the size of defensins (González et al., 2017). They have

361

a cysteine pairing pattern as vertebrate β-defensins (Rosa et al., 2011). Moreover, the N-

362

terminus comprises a parallel β-sheet and two α-helixes, and the C-terminus contains a

363

β-sheet as human β-defensin (Rosa et al., 2011). Due to these similarities, it has been

364

hypothesized that vertebrate β-defensins originated probably from an ancestral

365

invertebrate big defensin (Shafee et al., 2016; Tassanakajon et al., 2015; Zhu and Gao,

366

2013). N-terminus and C-terminus of big defensin have different antimicrobial

367

properties and inhibit gram-positive and gram-negative bacteria, respectively (Saito et

368

al., 1995).

369

3.2. Plants

heliomycin

from

Heliothis

11

virescens,

termicin

from

370

Most plant defensins are formed by a CSαβ motif, as invertebrate defensins (Figure 1).

371

However, most of the plant defensins are stabilised by four disulphide bridges. Base on

372

the precursor, plant defensins are classified in classes I and II. Class I does not have C-

373

terminal prodomain, while class II possesses a C-terminal prodomain that is cleaved to

374

release the mature defensin.

375

Several plant defensins have been isolated from seeds, and it has been proposed that

376

defensins could protect seed from soil microbes (Broekaert et al., 1995). Furthermore,

377

they have been identified in other tissues, such as leaves, flowers and roots. Defensins

378

can be produced in a organ-specific way, as is the case of NbDef1.1 in Nicotiana

379

benthamiana which is produced in leaves, stems, roots, seed and flowers; while

380

NbDef2.2 is only produced in flowers (Bahramnejad et al., 2009).

381

Plant defensins are potent antifungal agents. Typically, they interact with sphingolipids

382

in the fungal plasma membrane, and also they induce ROS formation and apoptosis

383

(Aerts et al., 2007; Thevissen et al., 2003; van der Weerden et al., 2008). Additionally,

384

some plant defensins exhibit insecticidal activity (Chen et al., 2002), and they

385

participate in fertilisation, flowering and zinc tolerance (Mirouze et al., 2006; Okuda et

386

al., 2009; Wilson et al., 2005).

387

The involvement of defensins and other AMPs in the stress response not related to

388

pathogens is not clear. Defensins might be involved in stress adaptation in addition to

389

the antimicrobial activity (Campos et al., 2018).

390

3.3. Fungi

391

Fungal defensins are classified according to their antimicrobial activity: antifungal or

392

antibacterial. Antifungal defensins contain β-sheets, and they interfere with cell wall

393

synthesis via binding to chitin (a primary component of the fungal cell wall) and

394

inhibition of chitin synthase III and V (Hagen et al., 2007). On the other hand,

395

antibacterial fungal defensins contain a CSαβ motif.

396

It has been demonstrated that some CSαβ defensins, α- defensins as well, inhibit the

397

synthesis of the bacterial cell wall via binding to lipid II, which is a peptidoglycan

398

precursor (de Leeuw et al., 2010; Schmitt et al., 2010; Schneider et al., 2010).

399 400

Conclusions

401

Both plants and animals have an innate defence mechanism that recognises and

402

responds to pathogens, and their defence response signalling pathways show some

403

similarities, such as the common presence of receptors containing leucine-rich. Once the 12

404

defence mechanism is activated, a response, which includes the expression of defensins

405

and other AMPs, is triggered. An organism produces several types of defensins. In

406

human, for example, to date, 26 defensins have been described. These defensins are not

407

regulated in the same manner and different signalling pathways regulate the gene

408

expression. Some defensins are constitutively expressed, as HBD-1 which is expressed

409

in keratinocytes. Other defensins are differentially regulated, as in the case of HD-5,

410

which is induced by bacterial in Paneth cells.

411

During infection, an orchestrated response activates the expression of various AMPs.

412

Besides defensins, animals produce other classes of AMPs, such as cathelicidins. Some

413

of them are regulated by the NF-κB, and the promoters show high similarities in their

414

transcription factor binding sites in comparison to defensins.

415

Beyond the direct attack against pathogens, novel functions have been reported for

416

defensins, such as immunomodulators and immune cell attractors. Likely many

417

functions of defensins remain uncertain. Understanding the transcriptional regulation

418

and their inducers could help us to elucidate new roles of defensins.

419

The emergence of multidrug-resistant (MDR) bacteria has become a global public

420

health concern. Antibiotic resistance is spreading faster than the introduction of new

421

antimicrobial compounds into the market. Therefore, the development of new effective

422

novel antibiotics is urgently needed, and AMPs are promising candidates (Lewies et al.,

423

2019). AMPs are potential agents against multi-drug resistant bacteria, but high

424

haemolytic activity and low salt -resistance are ones of the drawbacks for the clinical

425

applications of AMPs. Nevertheless, some defensins archive low hemolytic effect, even

426

up to 1024 µg/mL (Oeemig et al., 2012; Zhu et al., 2012). On the other hand, some

427

defensins, such as HBD-3 and plant defensins, exhibit a high salt-resistance (Harder et

428

al., 2001; Kerenga et al., 2019; Yang et al., 2018). Therefore, defensins are promising

429

therapeutic agents.

430

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Yang, M., Zhang, C., Zhang, M.Z., Zhang, S., 2018. Beta-defensin derived cationic antimicrobial peptides with potent killing activity against gram negative and gram positive bacteria. BMC Microbiol. 18, 54. https://doi.org/10.1186/s12866-018-1190-z. Yao, T., Lu, J., Ye, L., Wang, J., 2019. Molecular characterization and immune analysis of a defensin from small abalone, Haliotis diversicolor. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 235, 1-7. https://doi.org/10.1016/j.cbpb.2019.05.004. Yu, L.T., Xiao, Y.P., Li, J.J., Ran, J.S., Yin, L.Q., Liu, Y.P., Zhang, L., 2018. Molecular characterization of a novel ovodefensin gene in chickens. Gene 678, 233-240. https://doi.org/10.1016/j.gene.2018.08.029. Zasloff, M., 2002. Antimicrobial peptides of multicellular organisms. Nature 415, 389395. https://doi.org/10.1038/415389a. Zhang, G., Sunkara, L.T., 2014. Avian antimicrobial host defense peptides: from biology to therapeutic applications. Pharmaceuticals 7, 220-247. https://doi.org/10.3390/ph7030220. Zhang, L., Chen, D., Yu, L., Wei, Y., Li, J., Zhou, C., 2019. Genome-wide analysis of the ovodefensin gene family: monophyletic origin, independent gene duplication and presence of different selection patterns. Infect. Genet. Evol. 68, 265-272. https://doi.org/10.1016/j.meegid.2019.01.001. Zhang, L., Yang, D., Wang, Q., Yuan, Z., Wu, H., Pei, D., Cong, M., Li, F., Ji, C., Zhao, J., 2015. A defensin from clam Venerupis philippinarum: Molecular characterization, localization, antibacterial activity, and mechanism of action. Dev. Comp. Immunol. 51, 29-38. https://doi.org/10.1016/j.dci.2015.02.009. Zhang, R., Zhu, Y., Pang, X., Xiao, X., Zhang, R., Cheng, G., 2017. Regulation of antimicrobial peptides in Aedes aegypti Aag2 cells. Front. Cell Infect. Microbiol. 7, 22. https://doi.org/10.3389/fcimb.2017.00022. Zhao, B.C., Lin, H.C., Yang, D., Ye, X., Li, Z.G., 2016. Disulfide bridges in defensins. Curr. Top. Med. Chem. 16, 206-219. https://doi.org/10.2174/1568026615666150701115911. Zhao, C., Wang, I., Lehrer, R.I., 1996. Widespread expression of beta‐defensin hBD‐1 in human secretory glands and epithelial cells. FEBS Lett. 396, 319-322. https://doi.org/10.1016/0014-5793(96)01123-4. Zhao, J.G., Zhou, L., Jin, J.Y., Zhao, Z., Lan, J., Zhang, Y.B., Zhang, Q.Y., Gui, J.F., 2009. Antimicrobial activity-specific to Gram-negative bacteria and immune modulation-mediated NF-κB and Sp1 of a medaka β-defensin. Dev. Comp. Immunol. 33, 624-637. https://doi.org/10.1016/j.dci.2008.11.006. Zhao, J., Song, L., Li, C., Ni, D., Wu, L., Zhu, L., Wang, H., Xu, W., 2007. Molecular cloning, expression of a big defensin gene from bay scallop Argopecten irradians and the antimicrobial activity of its recombinant protein. Mol. Immunol. 44, 360-368. https://doi.org/10.1016/j.molimm.2006.02.025. Zhou, S., Ren, X., Yang, J., Jin, Q., 2018. Evaluating the value of defensins for diagnosing secondary bacterial infections in influenza-infected patients. Front. Microbiol 9, 2762. https://doi.org/10.3389/fmicb.2018.02762. Zhou, Y., Lei, Y., Cao, Z., Chen, X., Sun, Y., Xu, Y., Guo, W., Wang, S., Liu, C., 2019. A β-defensin gene of Trachinotus ovatus might be involved in the antimicrobial and antiviral immune response. Dev. Comp. Immunol. 92, 105-115. https://doi.org/10.1016/j.dci.2018.11.011. Zhu, S., Gao, B., 2013. Evolutionary origin of β-defensins. Dev. Comp. Immunol. 39, 79-84. https://doi.org/10.1016/j.dci.2012.02.011. Zhu, J., Wang, H., Wang, J., Wang, X., Peng, S., Geng, Y., Wang, K., Ouyang, P., Li, Z., Huang, X., Chen, D., 2017. Identification and characterization of a β-defensin gene 28

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involved in the immune defense response of channel catfish, Ictalurus punctatus. Mol. Immunol. 85, 256-264. https://doi.org/10.1016/j.molimm.2017.03.009. Zhu, S., 2008. Discovery of six families of fungal defensin-like peptides provides insights into origin and evolution of the CSαβ defensins. Mol. Immunol. 45, 828-838. https://doi.org/10.1016/j.molimm.2007.06.354. Zhu, S., Gao, B., Harvey, P.J., Craik, D.J., 2012. Dermatophytic defensin with antiinfective potential. Proc. Natl. Acad. Sci. USA 109, 8495-8500. https://doi.org/10.1073/pnas.1201263109. Zipfel, C., 2008. Pattern-recognition receptors in plant innate immunity. Curr. Opin. Immunol. 20, 10-16. https://doi.org/10.1016/j.coi.2007.11.003. Zou, J., Mercier, C., Koussounadis, A., Secombes, C., 2007. Discovery of multiple betadefensin like homologues in teleost fish. Mol. Immunol. 44, 638-647. https://doi.org/10.1016/j.molimm.2006.01.012.

29

1190

Figure captions

1191

Figure 1. Diversity of defensins. Phylogenetic tree of CSαβ defensins (green), α-

1192

defensins (red), β-defensins (black), β-defensin- from fish (orange) and big defensins

1193

(blue). The phylogenetic tree was constructed with the full-length amino acid sequences

1194

using the neighbour-joining method in MEGAX. Numbers on the branches indicate the

1195

bootstrap percentage values (1,000 replicates). The name of the defensin, origin and

1196

NCBI accession number of each sequence are indicated.

1197 1198

Figure 2. Representative structures of defensins based on their secondary structure.

1199

Defensins are classified into: (A) α- (HD-5 from human; protein data bank: 2LXZ), (B)

1200

β- (HBD4 from human; protein data bank: 5KI9), (C) θ- (RTD-1 from Rhesus macaque;

1201

protein data bank: 2LYF), and (D) cysteine-stabilised αβ (CSαβ) (AhPDF1 from

1202

Arabidopsis halleri; protein data bank: 2M8B). Disulphide bridges are displayed as

1203

orange lines. α- helices are represented in pink coiled ribbons, β- in yellow arrows, coils

1204

in white and 3-10 helix in blue. Structures were visualised using Visual Molecular

1205

Dynamics (VMD) (Humphrey et al., 1996).

1206 1207

Figure 3. Simplified schematic overview of the defence signalling pathway that triggers

1208

the expression of defensin in mammals, insects (Drosophila) and plants (Arabidopsis).

1209

A) In mammals, pathogen-associated molecular patterns (PAMPs) are recognised by

1210

pattern recognition receptors (PRR). This interaction activates MyD88 which triggers a

1211

signalling cascade (dashed lines) which results in the nuclear translocation of NF-κB

1212

transcriptional factors. These transcriptional factors regulate the defence response that

1213

includes the expression of defensins and other AMPs. Cytokines also can trigger and

1214

activate NF-κB. B) In insects, the innate immune system involves two main signalling

1215

pathways: Imd and Toll, whose transcriptional factors are Relish and DIF/Dorsal,

1216

respectively. C) In plants, bacterial and fungal infection activate two main responses,

1217

effector-triggered immunity (ETI) and MAMP-triggered immunity (MTI), both activate

1218

the MAPK cascade which activates the expression of defensins. Defensin gene

1219

expression also is triggered by plant hormones, such as jasmonic acid (JA), ethylene

1220

(ET) and salicylic acid (SA).

1221 1222

Figure 4. Localisation of the gene expression of defensins in mammals, insects and

1223

plants. Defensins can be constitutively or differentially expressed in vertebrates, 30

1224

invertebrates and plants. An example of each group is represented in mouse (mammal,

1225

A), Drosophila (insect, B) and Arabidopsis (C).

31

Table 1. Transcriptional expression of human defensin genes.

Defensin name (gene)

mRNA distribution Constitutive expression

Reference Inducible expression Stimulator Localisation

α-defensins HD-5 ( DEFA5)

Toxoplasma gondii, IFN-β, Unmethylated CpG

HD-6 ( DEFA6)

β-defensins HBD-1 (DEFB1)

HBD-2 (DEFB2)

Paneth cell Paneth cell

Keratinocytes, airway epithelia, epithelia from the urogenital tract, monocytes, macrophages. Macrophages, monocytes, airway epithelia

LPS, IFN-γ

Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, Candida albicans, TNF-α, IL-1β, 1L-17.

Monocytes, macrophages, dendritic cells. Keratinocytes, airway epithelia, intestinal tract, monocytes, macrophages. Keratinocytes, airway epithelia

(Duits et al., 2002; Zhao et al., 1996)

(García et al., 2001; Premratanachai et al., 2004; Yamaguchi et al., 2002) (Yamaguchi et al., 2002; Zhou et al., 2018) (Kao et al., 2003; Yamaguchi et al., 2002) (Premratanachai et al., 2004)

HBD-3 (DEFB3)

Keratinocytes

Pseudomonas aeruginosa, IFN-γ

HBD-4 (DEFB4)

Epididymis, gingival keratinocytes

Streptococcus pneumoniae, GramPseudomonas aeruginosa

Airway epithelia

HBD-5 (DEFB5)

Epididymis

H7N9 virus

Blood

HBD-6 (DEFB6)

Testis, lung, epididymis

HBD-7 ( Beta-defensin 107)

Gingival keratinocytes

32

(Foureau et al., 2010; Santamaria et al., 2016)

(Harder et al., 1997; Harder et al., 2000; Kao et al., 2004; Krisanaprakornkit et al., 2000; Liu et al., 2002) (Fahlgren et al., 2004; Harder et al., 2001)

HBD-8 (DEFB108)

Testis

Candida sp.

HBD-9 (DEFB109)

Aspergillus fumigatus

HBD-11 (DEFB111)

Heart, brain, placenta, lung, liver, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, leukocyte, ocular surface, gingival keratinocytes Gingival keratinocytes

HBD-12 (DEFB112)

Gingival keratinocytes

HBD-14 (DEFB114)

Gingival keratinocytes Bronchial epithelial

(Kao et al., 2003; Premratanachai et al., 2004) (Abedin et al., 2008; Alekseeva et al., 2009; Kao et al., 2003; Premratanachai et al., 2004) (Premratanachai et al., 2004) (Premratanachai et al., 2004)

Candida sp.

Gingival keratinocytes

(Premratanachai et al., 2004)

HBD-18 (DEFB118)

Pancreas, testis

(Kao et al., 2003)

HBD-19 ( DEFB119)

Testis

(Djureinovic et al., 2014)

HBD-21 (DEFB121)

Testis

(Fagerberg et al., 2014)

HBD-23 (DEFB123)

Testis

(Fagerberg et al., 2014)

HBD-25 (DEFB125)

Testis, skeletal muscle, kidney

HBD-26 ( DEFB126)

Prostate, testis, skeletal muscle, pancreas

HBD-27 (DEFB127)

Testis, kidney, pancreas, skeletal muscle

HBD-28 (DEFB128)

Testis and epididymis

HBD-29 (DEFB129)

Testis, skeletal muscle

(Fagerberg et al., 2014; Rodríguez-Jiménez et al., 2003) (Fagerberg et al., 2014; Rodríguez-Jiménez et al., 2003) (Fagerberg et al., 2014; Rodríguez-Jiménez et al., 2003; Zhou et al., 2018) (Rodríguez-Jiménez et al., 2003) (Kao et al., 2003; RodríguezJiménez et al., 2003)

33

Acinetobacter baumannii

Blood

HBD-31 (DEFB131)

34

Prostate, testis, small intestine

(Kao et al., 2003)

Table 2. Transcriptional expression of fish defensin genes.

mRNA distribution Inducible expression Species Rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss)

Defensin gene omDB-1

Muscle, head kidney

omDB-1

Skin, spleen, gut, gill, head kidney, liver

omDB-2

omDB-3

omDB-4 Common carp (Cyprinus carpio) Gilthead seabream (Sparus aurata)

BD1 BD2 saBD β-defensin

35

Stimulator

Constitutive expression

Skin, spleen, gonad, gut, gill, head kidney, liver Skin, spleen, gonad, gut, gill, head kidney, liver, brain

Response

Reference (Falco et al., 2008)

Yersinia ruckeri

Skin (-), gill (-), gut (-)

polyI:C

Head kidney primary cell culture (↑)

Y. ruckeri

Skin (-), gill (-), gut (-)

polyI:C

Head kidney primary cell culture (-)

Y. ruckeri

Skin (-), gill (↑), gut (-)

polyI:C Y. ruckeri

Skin, spleen, gonad, gut, gill, head kidney, liver

polyI:C

Skin Liver Skin, peritoneal leucocytes, head kidney, liver, gonad, gut, gill, spleen, brain, thymus

β -Glucan β -Glucan Unmethylated CpG oligodeoxynucleotides, bacterial DNA

Head kidney, intestine

Nannochloropsis gaditana

Head kidney primary cell culture (↑) Skin (-), gill (-), gut (-) Head kidney primary cell culture (-) Skin (↑) Skin (↑), gill (↑) Head-kidney leucocytes in vitro (↑) Head kidney (↑)

(Casadei et al., 2009)

(van der Marel et al., 2012) (Cuesta et al., 2011) (Cerezuela et al., 2012)

Olive flounder (Paralichthys olivaceus)

fBDI

Larvae

Edwardsiella tarda

Head kidney (↑)

Medaka (Oryzias latipes)

β-defensin

Eyes, liver, kidney, blood, spleen, gill, intestine.

LPS

Eyes (↑)

β-defensin

Pituitary, testis

EcDefensin

Liver, skin, gill, muscle, spleen, kidney, brain, intestine, heart, stomach, head kidney

LPS, Singapore grouper iridovirus, polyI:C

Spleen (↑), liver (↑)

(Guo et al., 2012)

maΒD-1

Skin, blood, liver, kidney, gill, hindgut

Aeromonas sobria

Liver (↑), skin (↑), gill (-)

(Liang et al., 2013)

A. sobria

Liver (↑), skin (↑), gill (↑), foregut (↑)

(Liang et al., 2013)

Orange-spotted grouper (Epinephelus coioides)

Blunt snout bream (Megalobrama amblycephala) Blunt snout bream (Megalobrama amblycephala)

36

maΒD-2

Liver, kidney, brain, foregut Heart, brain, intestine, liver, gill, head kidney, trunk kidney, spleen, muscle Eye, muscle, brain, skin and hindgut, heart and ovary, spleen, kidney, gill, liver

Mandarin fish (Siniperca chuatsi)

ScBD

Chinese loach (Paramisgurnus dabryanus)

pdBD

Soiny mullet (Liza haematocheila)

Lhβdefensin

Spleen, kidney, gut, liver

Streptococcus dysgalactiae

Channel catfish (Ictalurus punctatus)

Edwardsiella ictaluri

ccBD

Skin, stomach, spleen, kidney, head kidney, liver, foregut, hindgut, gill

Nile tilapia (Oreochromis niloticus)

Onβdefensin

Atlantic cod (Gadus morhua)

defb

Skin, spleen, kidney, muscle, liver, heart, intestine, stomach, gill Swim bladder, peritoneum

(Nam et al., 2010)

(Zhao et al., 2009) (Jin et al., 2010b)

(Wang et al., 2012) Aeromonas hydrophila

Eyes (↑), gill (↑), skin (↑), spleen (↑)

(Chen et al., 2013)

Spleen (↑), gut (↑), kidney (↑), liver (↑) Head kidney (↑), gill (↑), skin (↑), spleen (-) Spleen leucocytes (↑), Head kidney leucocytes (↑)

(Zhu et al., 2017) (Zhu et al., 2017)

Streptococcus agalactiae

Skin (↑), muscle (↑), kidney (↑), gill (↑)

(Dong et al., 2015)

Vibrio anguillarum

Head kidney (↑)

(Ruangsri et al.,

LPS

(Qi et al., 2016)

wall, skin, head, gill and kidneys

2013) Temperature increase Commensal bacterium Pseudomonas sp. LPS

Meagre (Argyrosomus regius)

defb

Kidney, spleen, gut, gill

poly (I:C) β-glucan

Spleen, gills, brain, skin, hindgut, liver, muscle, foregut, buccal epithelium, blood, head kidney, gonad Gill, gonad, gut, kidney, muscle, skin, liver, spleen. (zfDB2 expressed only in gut)

Gills (↓), skin (↑)

(Campoverde et al., 2017)

Hind-gut (↑), Spleen (↑), gills (↑), skin (↑), foregut (↑), head kidney (↑)

(Li et al., 2014)

β-defensin 3

Zebrafish (Danio rerio)

zfDB1, zfDB3

Rainbow trout (Oncorhynchus mykiss)

omDB-1-4

Skin, gill, gut, liver

Peptidoglycan

Dabry's sturgeon (Acipenser dabryanus)

AdBD

Skin, liver, gonad, muscle, brain, eye, head kidney, heart, hind gut, spleen, gill

E. tarda

Grass carp (Ctenopharyngodon idella)

β-defensin1

Intestine

Gossypol

Intestine (↓)

Golden pompano (Trachinotus ovatus)

Head kidney, spleen, brain, muscle, skin, heart, gill, liver, stomach, intestine

Vibrio harveyi

Head kidney (↑), spleen (↑)

TroBD

Viral nervous necrosis virus

Head kidney (↑), spleen (↑)

PolyI:C: polyribocytidylic acid, a synthetic dsRNA. LPS: lipopolysaccharide.

V. anguillarum

(Ruangsri et al., 2014)

Kidney (↑), spleen (↓), gut (↓), gill (↓) Kidney (-), spleen (↓), gut (-), gill (-) Kidney (↑), spleen (-), gut (-), gill (-)

Common carp (Cyprinus carpio)

Not effect (-), up-expressed (↑), down-expressed (↓).

37

Gills (↓), skin (↓)

(Zou et al., 2007) Skin (↑), gill (↑), gut (↓), liver (↓) Hind gut (↑), Skin (↑), gill (-), liver (-), head kidney (↑), spleen (↑)

(Casadei et al., 2013) (Chen et al., 2019) (Wang et al., 2019) (Zhou et al., 2019)

Table 3. Transcriptional expression of invertebrate defensin genes. Species

Oyster (Crassostrea gigas)

Defensin gene

Cg-Def

Cg-defh2

Soft tick (Ornithodoros moubata)

Cg-BigDef1, Cg-BigDef2, Cg-BigDef3 Defensins A and B Defensins C and D

Disk abalone (Haliotis discus discus) Mediterranean mussel (Mytilus galloprovincialis)

defensin

Migratory locust (Locusta migratoria)

LmDEF1

MGD-1 MgBD1 MgBD3 MgBD6

LmDEF3 LmDEF5 Mosquito (Aedes aegypti)

38

DefA, DefB, DefC

Constitutive expression Mantle, posterior abductor muscle, labial palps, hemocytes Hemocytes Hemocytes, gonad, digestive gland, gills Midgut Midgut, fat body and reproductive tract Mantle, hepatopancreas Hemocytes Digestive gland Digestive gland Digestive gland, mantle, posterior abductor muscle

Fat body, salivary glands Callow pupae

mRNA distribution Inducible expression Stimulator Response Micrococcus luteus, Vibrio splendidus, Mantle (-) Vibrio anguillarum

Reference

(Gueguen et al., 2006)

M. luteus, V. splendidus, V. anguillarum

Hemocytes (↓), Mantle (↑), gills (↑)

(Gonzalez et al., 2007)

V. splendidus, Vibrio tasmaniensis

Hemocytes (↑)

(Rosa et al., 2011)

Escherichia coli, Staphylococcus aureus, blood feeding Blood feeding

Midgut (↑)

(Nakajima et al., 2001) (Nakajima et al., 2002)

Vibrio alginolyticus, Vibrio parahemolyticus, Lysteria monocytogenes

Hemocytes (↑), gills (↑) digestive tract (↑)

(De Zoysa et al., 2010)

V. alginolyticus

Hemocytes (↓)

(Mitta et al., 1999) (Gerdol et al., 2012)

Metarhizium anisopliae Nosema locustae Metarhizium anisopliae, N. locustae N. locustae

Fat body (↑), salivary glands (↑) Salivary glands (↑) Fat body (↑), salivary glands (↑) Salivary glands (↑)

(Lv et al., 2016) (Lv et al., 2016) (Lv et al., 2016) (Lv et al., 2016)

E. coli, M. luteus

Fat body (↑)

(Lowenberger et al., 1999a; Ramirez et al., 2018)

Midgut (↑)

DefA, DefB

DefC

Drosophila sp.

Drosomycin

Defensin

Termite (Pseudacanthotermes spiniger) House fly (Musca domestica)

Termicin

Md-defensin (CSαβ)

Midgut

Salivary glands, spermathecae and seminal receptacle

Larval antennomaxillary organ, labellar glands, spermathecae and seminal receptacle Hemocytes and salivary glands Epidermis of the body wall, hemocytes, larvae.

E. coli, Serratia marcescens, Staphylococcus aureus, Enterococcus faecium, Leucobacter spp., Candida albicans). Beauveria bassiana, Isaria javanica

Aag2 cells (↑)

(Zhang et al., 2017)

Midgut (↑), fat body (↑)

Plasmodium gallinaceum

Midgut (-)

Beauveria bassiana, I. javanica

Midgut (↑), fat body (↑)

P. gallinaceum

Midgut (-)

M. luteus, E. coli, Erwinia carotovora carotovora

Fat body (↑), Tracheal system (↑)

Saccharomyces cerevisiae

Larval salivary gland (↑), fat body (↑) Fat body (↑)

(Ramirez et al., 2018) (Lowenberger et al., 1999b) (Lowenberger et al., 1999b; Ramirez et al., 2018) (Lowenberger et al., 1999b) (Fehlbaum et al., 1994; Ferrandon et al., 1998) (Seto and Tamura, 2013) (Dimarcq et al., 1994; Tzou et al., 2000) (Seto and Tamura, 2013) (Lamberty et al., 2001; Liu et al., 2015) (Wang et al., 2006)

M. luteus, E. coli

S. cerevisiae Metarhizium anisopliae

E. coli Bacillus thuringiensis Injury

Triangle-shell pearl mussel (Hyriopsis cumingii)

39

HcDef1 HcDef2

Hepatopancreas, gills Hepatopancreas, gills

Larval salivary gland (↑), fat body (↑) Adults (↑)

Fat body (↑), epidermis of the body wall (↑) Midgut (↑) Fat body (↑), hemocytes (↑), larvae (↑), pupae (↑) Gills (↓)

V. anguillarum Gills (↓)

(Mura and Ruiu, 2017) (Andoh et al., 2018) (Ren et al., 2011)

HcDef3 HcDef4 HcDef5 HcDef6 Manila clam (Ruditapes philippinarum) Zebra mussel (Dreissena polymorpha)

Rpdef1, Rpdef2, Rpdef3, Rpdef4 Dpd

Clam (Venerupis philippinarum)

VpDef

Scallop (Argopecten irradians)

AiBD

Scallop (Argopecten purpuratus)

ApBD1

Noble scallop (Chlamys nobilis)

CnBD

Triangle-shell pearl mussel (Hyriopsis cumingii) Horseshoe crab (Tachypleus tridentatus)

HcBD

Big defensin

Japanese spiny lobster (Panulirus japonicus)

PjD1, PjD2

Small abalone (Haliotis

HdDef-2

40

Hepatopancreas, gills Hepatopancreas, gills Gills Gills Hemocytes, mantle, gills, foot, digestive gland Foot, ctenidium, mantle, hemocytes, muscle Hemocytes, mantle, gills, hepatopancreas

V. anguillarum

V. anguillarum

Hemocytes (↑)

(Zhang et al., 2015)

Haemocytes, gills

V. anguillarum

Haemolymph (↑)

(Zhao et al., 2007)

Vibrio splendidus

Hemocytes (-), mantle (-), gills (↑)

(González et al., 2017)

Vibrio parahaemolyticus

Hemocytes (↑)

(Yang et al., 2016)

Aeromonas hydrophila

Mantle (↑), liver (↑), intestine (↑), gill (↑), foot (↑)

(Wang et al., 2014)

Mantle, muscle, gills, digestive gland, gonad Gonad, mantle, gill, hemocytes, intestine, adductor, muscle Mantle, blood, liver, kidney, intestine, stomach

Gills (↓) Gills (↑) Gills (↓) Gills (↑) Hemocytes (↑)

(Xu and Faisal, 2010)

Hemocytes Heart, nerves, intestine, hemocytes, gills, hepatopancreas Mantle, gill, hepatopancreas,

(Wang et al., 2015b)

Saito et al., 1995

Pisuttharachai et al., 2009 Vibrio harveyi

Hepatopancreas (↑)

Yao et al., 2019

diversicolor) foot Not effect (-), up-expressed (↑), down-expressed (↓).

41

Table 4. Some plant defensins and their transcriptional expression. Species

Defensin gene

mRNA distribution Inducible expression

Constitutive expression

Stimulator Rice (Oryza sativa)

Arabidopsis thaliana

OsDEF7

Germinating seed

OsDEF8

Flower

PDF1.1 PDF1.2

Seed, siliques Rosettes

PDF2.1

PDF2.2 PDF2.3

Tabaco (Nicotiana benthamiana)

NbDef1.1

NbDef2.2 Wheat (Triticum aestivum L.)

42

Tad1

References Response

Xanthomonas oryzaepv.oryzae Imbibition, anoxia Xanthomonas oryzaepv.oryzae Cold, dehydration Alternaria brassicicola Ethylene, methyl jasmonate, oxidative stress, silver nitrate, A. brassicicola, Fusarium oxysporum f. sp. Matthiolae

Leaves (↑) Embryo (↑) Leaves (↑) Seedling (↑), leaves (↑) Leaves (↑) Leaves (↑)

Silique

Heterodera schachtii

Roots (↑)

Root, flower, silique, leaves Siliques, flowers, stems, leaves

Wounding A. brassicicola

Leaves (↑) Leaves (↓)

Leaves, stems, roots, flowers, developing seeds Flower

Pseudomonas syringae wounding, ethylene Cold, high salinity

pv.

Tabaci,

Leaves (↑) Shoot (↑)

(Weerawanich et al., 2018)

(Tesfaye et al., 2013) (Epple et al., 1997; Penninckx et al., 1996; Tesfaye et al., 2013) (Siddique et al., 2011; Thomma et al., 1998) (Thomma et al., 1998) (Epple et al., 1997)

(Bahramnejad et al., 2009) (Koike et al., 2002)

Pepper (Capsicum annuum)

CADEF1

Stem, root green fruit

and

Tomato Tgas118 Anthers, petals and (Lycopersicon pistil esculentum) Not effect (-), up-expressed (↑), down-expressed (↓).

43

X. campestris pv. Vesicatoria, salicylic acid, methyl jasmonate, abscisic acid, hydrogen peroxide, wounding, high salinity, drought, benzothiadiazole, D, L-β-amino-n-butyric acid

Leaves (↑)

(Do et al., 2004)

Gibberellin, wounding, dehydration

Leaves (↑)

(van den Heuvel et al., 2001)

Table 5. Fungal defensins characterised regarding their gene expression.

44

Species Aspergillus nidulans

Fungal defensin CSαβ defensin Anisin

Stimulator

Aspergillus clavatus

Aclasin

Aspergillus giganteus

Defensin-like antifungal AFP

Aspergillus niger

AnAFP

Carbon starvation (↑), alkaline pH (↑) acid pH (↓), heat shock (↑), osmotic stress (↑), ethanol (↑), fungal presence (↑) Carbon starvation (↑)

Penicillium chrysogenum Penicillium digitatum

PAF

Carbon starvation (↑)

AFPB

Rich medium (↓)

Heat shock (↑) Oxidative stress (↑), Gram+ bacteria (Bacillus megaterium)(↑)

References (Eigentler et al., 2012) (Contreras et al., 2019)

(Meyer and Stahl, 2003; Meyer et al., 2002) (Paege et al., 2016) (Marx, 2004) (Garrigues et al., 2016)

A

B

C

C

C

D

N CC

CC

CC CC CC

N

C

CC

N

CC CC CC

CC CC CC

N CC

C

Mammals

A Injury Infection

Bacterial and fungal infection

Cytokines

PAMPs

C

Insects

B

Infection Avr

PAMPs

Spaetzle Toll

TLR

FLS2 MAPK pathway Tube/dMyD88

IKK

Injury Infection Abiotic stress

Bacterial and fungal infection

Bacterial infection

PGRP-LC IL-1R

Plants

MTI

MEKK

R

ETI

Imd

MyD88

DmIKK MKK4/MKK5

IκB

Rel-49

NF- κB Gene expression of defensins and other AMPs

Relish

IMD pathway

Cactus

DIF/Dorsal MPK3/MPK6

Toll pathway Gene expression of defensins and other AMPs

WRKY

Gene expression of defensins and other AMPs

A

Mammals

Insects

B

Epithelial cells in respiratory, reproductive and digestive tract.

Epithelial cells in skin, respiratory, reproductive, and gastrointestinal tract.

C

Plants Seeds and flowers

Leaves Roots Fat body Neutrophils, macrophages, monocytes

Hemocytes

Highlights Defensins are one the largest group of antimicrobial peptides. They have a broad spectrum of antimicrobial activity. In animals and plants, defensins can be constitutively or differentially expressed. Conserved signalling pathways regulate the transcription of defensin genes.